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Mammoth trouble

By Philip Cohen

A POPULAR type of DNA fingerprinting used by conservation biologists may give
false results with some species of animals, say researchers in Germany.

There are two sources of DNA in animals: the nucleus and the energy-producing
powerhouses of the cell called the mitochondria. Mitochondrial fingerprinting is
normally used when the genetic material in a sample is degraded, for while there
are only two copies of each nuclear gene in a cell, there are often hundreds of
copies of mitochondrial DNA (mtDNA).

In these cases, a technique called polymerase chain reaction, or PCR, is used
to amplify undamaged strands of mtDNA so that they can be analysed. PCR analysis
of mtDNA is increasingly used by field biologists to track the relationships or
numbers of animals in the wild. It is also used by forensic experts to track
criminals.

But Alex Greenwood and Svante Pääbo of the University of Munich
came across a potential flaw in the fingerprinting technique while studying
woolly mammoths. The team wanted to use tissue from hundreds of mammoth
carcasses found in permafrost to work out the relationships among the animals
and compare them to their distant cousins, modern elephants.

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To start with, they took hairs from an Asian elephant (Elephas
maximus) at a nearby zoo and amplified a fragment of mtDNA. But when they
tried to sequence the products of the PCR, they found they were scrambled. “So
we suspected we were dealing with a mixture,” says Pääbo. The
suspicion was confirmed when they purified and sequenced individual DNA strands
from the PCR. They found four different classes of sequence.

When the researchers looked at elephant blood, however, they found only one
of the four sequence classes found in the hair. They also discovered that it was
the only active sequence of mtDNA, meaning that the other three found in the
hair were fragments of mtDNA that had migrated to the nucleus early in
evolution. This suggests that elephant hair has few mitochondria, giving a high
chance that PCR would amplify one of these “pseudogenes”, rather than the true
sequence. Pääbo points out that if no blood had been available for
analysis—as is often the case in forensics or molecular ecology—it
may have been impossible to sort out the true sequence.

Mitchell Holland, a DNA identification expert at the Armed Forces Institute
of Pathology in Rockville, Maryland, says the finding highlights the great care
that must be taken in the analysis of mtDNA data(see “Ghosts in the machines”).
“You can’t just get a PCR result and walk away from it,” he says. But he
adds that nuclear pseudogenes are not a problem in human hair analysis. He says
that researchers can recognise the sequences of mtDNA that have migrated to the
nucleus, and that they rarely see them in their PCR reactions.

Pääbo agrees that “contaminating” nuclear mtDNA sequences are
probably only an issue for particular tissues in particular animals. “Most
sequences out there are probably fine,” he says. “This is just a warning to
people that you have to be aware of this possibility and be really careful.” But
his plans to map familial relationships in mammoths seem to have been scuppered.
PCR amplification of mtDNA from the hair of one of these ancient beasts reveals
a variety of mitochondrial sequences as confusing as that in E. maximus.